3625-32000-104农业和食品研究计划竞争性赠款号:研究是由美国农业部ARS克里斯支持。 2011-67015-30736来自粮食和农业的美国农业部研 ​​究所。我们感谢杰西卡·波洛克，艾玛Frimml -摩根，雪莱齐默尔曼，克里斯汀低音，布鲁斯·佩施，莫莉Stafne，艾伦詹森和特雷西·波特的出色的技术援助以及丽贝卡麦迪逊，道格·尤因，凯蒂Pille先生，周杰伦斯特芬，大卫·吕贝尔斯，罗宾Zeisness，和大卫Panthen动物的良好的护理和处理。

1.准备了以下解决方案<ol><li>完全RPMI（cRMPI）加入胎牛血清（FBS）中至浓度为10％（体积/体积），谷氨酰胺（2μM），丙酮酸钠（1μM），非必需氨基酸（0.1μM），青霉素制备-streptomycin（100单位/ ml青霉素和0.1mg / ml链霉素）和2-巯基乙醇（50毫摩尔）进RPMI 1640中。 </li><li>制备2×柠檬酸葡萄糖，通过混合柠檬酸钠（77μM），柠檬酸（38μM），和右旋糖（122μM），在蒸馏水中。 </li><li>制备的Ca 2+镁离子的磷酸盐缓冲盐水（PBS）pH 7.2的:氯化钠（137毫米），氯化钾（2.7毫摩尔），的Na 2 HPO 4（二元，10毫摩尔），KH 2 PO 4（一元，2毫摩尔）在蒸馏水中;将pH调节至7.2。 </li><li>通过加入牛血清白蛋白的PBS（体积/重量，1G / 100毫升的PBS）制备PBS中的1％（PBS中的1％BSA）中。 </li><li>制备PBS + 0.05％吐温20（PBST）中加入0.05％吐温20的PBS（体积/体积，500微升吐温20℃/ 1升PBS中）。 </li><li>制备100毫摩尔Tris HCL pH值为8.2的缓冲通过在蒸馏水中混合三（100毫米），盐酸（0.50毫摩尔）。 </li><li>通过在蒸馏水中稀释的乙醇（纯度≥99.5％）（体积/体积，34毫升甲酸乙酯乙醇/ 100毫升蒸馏水）制备35％的乙醇溶液。 </li><li>过滤消毒cRPMI，2×酸柠檬酸葡萄糖，PBS，PBS使用0.22微米的过滤器1％BSA。 </li></ol> 2.长期培养细胞（14日协议） （第一天） <ol><li>制备抗原溶液在cRPMI中的最终浓度的两倍。抗原的选择是至关重要的，应根据研究的目的。 </li><li>淡化重组M.结核抗原TB10.4和抗原Ag85A双至2微克/毫升（每抗原;终浓度为1微克/毫升）。 </li><li>稀M.牛纯蛋白衍生物（PPD-B，Prionics公司），以10微克/毫升（最终浓度为5微克/毫升）。 </li><li>稀释重组早期分泌抗原靶6（ESAT-6）和培养滤液蛋白10（CFP-10）的融合蛋白（雷沙特-6:CFP10的）至2微克/毫升（1微克/毫升的最终浓度）。 注意:这些抗原是免疫的IFN-γ诱导结核分枝杆菌的抗原和可被包括，以刺激的PBMC从M.牛感染的动物。卡介苗或M.牛 ΔRD1接种动物不响应雷沙特-6:CFP10，如在这些菌株被删除该区域编码这些抗原（即，RD1）。雷沙特-6:在这些研究中所用CFP10是从C爪牙博士，美国爱荷华州立大学的一种恩赐。 TB10.4和抗原Ag85A双是剧毒和疫苗M.目前免疫抗原牛 37株。 PPDB，作为纯化的蛋白衍生物，是数种抗原包括与非结核性分枝杆菌共享的抗原的复合物。 Infec泰德动物将有可能回应所有抗原（即:TB10.4，银85A，雷沙特-6:CFP10和PPDB），而接种疫苗的动物应不雷沙特-6回应:CFP10 11。 </li><li>板500微升/在一式四份抗原溶液在24孔板每个动物福利。对于这个协议，使用抗原作为池;因此，使用所有的孔包含所有的抗原和不管制（如空或有丝分裂原）这一步。在39°C / 5％的CO 2孵育板。牛的正常体温是39℃;因此，一些研究者利用39℃的牛细胞培养。此外，在与人体细胞某些情况下，在39℃的细胞培养提供了额外的好处26,30。 </li><li>再加上16或18G的注射针头用6ml的2×酸 - 柠檬酸盐 - 葡萄糖预负荷注射器;并收集将60ml牛血静脉穿刺颈。 </li><li>隔离的PBMC通过外周血血沉棕黄层组分的标准密度梯度离心所描述的毛厄等调节细胞浓度至4×10 6个细胞/ ml。27 </li><li>添加细胞（500微升/孔）到抗原预装24孔板，一式四份每只动物（步骤2.5）。在39°C / 5％的CO 2孵育板。 </li></ol> （天三套和七） <ol start="9"><li>使用无菌技术，小心地从每个孔中取出500μl的上清液，而不会干扰细胞层。 </li><li>通过加入500微升/孔的cRPMI含IL-2补充孔体积（30U /微升， 即 ，15单位/孔;重组人IL-2西格玛I7908）。在39°C / 5％的CO 2孵育板。 </li></ol> （天数10和12） <ol start="11"><li>使用无菌技术，从每个孔取出750微升上清液。补充750微升/ cRPMI的孔的体积（无IL-2）。在39°C / 5％的CO 2孵育板。 </li></ol><p类="jove_title"&gt; 3。酶联免疫斑点法（第三天协议） （第一天（长期协议，培养第 12天）） <ol><li>正确标注的ELISPOT板为每个组样品，电镀细胞时，为了避免错误:长期细胞加抗原呈递细胞（APC），长期细胞无APC和离体 /短期细胞（ 图1）。复制为每个治疗（即抗原刺激）应该至少一式两份进行。 </li><li>通过稀释的小鼠抗牛IFN-γ抗体（MCA2112，克隆CC330）的PBS（8微克/毫升）制备的捕获抗体溶液。 </li><li>使用多通道移液器预湿ELISPOT板的孔用15微升/孔的35％乙醇1分钟。为防止膜破损，不要触摸井用吸头的底部在任何时候在试验过程中。 </li><li>洗涤板6次，用PBS以300μl/孔。洗涤液应在板反转丢弃。洗涤应迅速进行，而不是让板孔干燥。 </li><li>捕获的抗IFN-γ抗体（来自上述步骤1）的吸移管100μl/孔。孵育在4°CO / N（保持板内的拉链储物袋）。 </li></ol> （二日（长期培养协议的第 13天）） <ol start="6"><li>制备抗原溶液在最终浓度的两倍。理疗:无刺激（cRPMI）的PPD-B（20微克/毫升，终浓度:10微克/毫升），TB10.4蛋白鸡尾酒和Ag85A双（每种蛋白的2微克/毫升，最终浓度:1微克/ ml的每种蛋白），雷沙特-6:CFP10的（对于牛枝原体感染，2微克/毫升，最终浓度:1微克/毫升），和阳性对照，如美洲商陆抗（20微克/毫升，终浓度:10微克/毫升）。可以用来代替PWM（ 如伴刀豆球蛋白A）其它有丝分裂原。 注:此步骤组成的抗原四种不同的治疗方法，是n使用催产素作为池（如在步骤2.5）。这四个理疗:NS，PPDB，蛋白质鸡尾酒（TB10.4 Ag85A双构成一个处理）和PWM。 </li></ol> 4.短期细胞培养和细胞粘附（装甲运兵车）隔离<ol><li>收集60毫升血液从采血月1日（下:长期培养14日实验方案）相同的动物，像以前孤立的PBMC。 </li><li>调节细胞浓度至2×10 6个细胞/ ml。标签管显然是防止错配电镀细胞时。这些细胞将被用于的APC隔离，也可以作为短期细胞培养（步骤6.5）。标签管与两个动物数目和文化的类型（在这种情况下: 体外 ，短期或新鲜细胞）。 </li><li>通过板块反转从酶联免疫斑点去除多余的捕获抗体。洗板6次用300μlPBST。 </li><li>除去尽可能多的PBST越好。吸管将50μl细胞悬浮液，以标记为长期细胞加的APC的孔中。阻止其他WEL的ls与cRPMI的200μl/孔。孵育90分钟或39°C。前期预热cRPMI或39°C（必要的后续步骤）。不用于贴壁细胞（APC细胞）分离新鲜的PBMC，将需要在后续步骤中，并应被存储。 </li></ol> 5.培养细胞<ol><li>在90分钟温育（步骤4.4，短期培养和贴壁细胞分离步骤）中，收获的长期培养的细胞。 </li><li>用5或10微升的移液管，与细胞上下吸管媒体分离细胞，结合一式四份复制来自每个动物成一个单一的15ml试管中。离心管5分钟，400g。 </li><li>离心后，弃去上清液通过管倒置。去除细胞沉淀。加入5毫升的PBS中，轻轻重新悬浮沉淀，如果需要的话。重复离心。重复洗涤步骤两次。由管倒置弃上清，轻轻地重新悬浮细胞1毫升cRPMI。调整细胞浓度到2×10 5个细胞/ ml。 </li></ol> 6.电镀新鲜外周血单个核细胞培养<ol><li>在90分钟温育后，摇上板振荡器平板30秒。由板倒置除去非粘附细胞。吸管150微升/暖cRPMI的井（从步骤4中，下:短期细胞和贴壁细胞（APC细胞）分离步骤）。 </li><li>摇板和丢弃洗涤液。重复洗涤3次。添加100μl/孔的每种抗原到合适的孔（预标记的）。 </li><li>板块细胞，吸移管100微升/孔的长期培养的细胞悬浮液（2×10 5个细胞/ ml）到标记为长期细胞加的APC的孔中。确保在此步骤中加入的长期细胞来自同一动物的APC是已经在井内。不要从不同动物的混合APC的和长期培养的细胞。 </li><li>长期培养的细胞悬浮液的吸移管100μl/孔（2×10 5个细胞/ ml）成孔未经APCS代表评估APC要求的长期培养的反应。 </li><li>添加100μl/孔的短期细胞（新鲜分离并调节至2×10 6个细胞/ ml）用于离体反应评估。孵育板O / N在39°C / 5％ 二氧化碳培养箱。确保板平放，而不要堆放板。 注意:如果细胞表型的分析是需要的，流式细胞仪可以作为辅助测定来进行。将细胞接种在96如对于ELISPOT测定孔U底板（代替ELISPOT平板）。捕获抗体吸附（步骤3.4至3.5）应该被跳过。然后将细胞温育O / N在39℃/ 5％CO 2，并进行了标准细胞染色协议。细胞染色试剂都包括在试剂表。 </li></ol>第三天（长期培养协议的第 14天） <ol start="6"><li>稀释的检测抗体（小鼠抗牛IFN-γ，克隆CC302）至5微克/毫升在PBS中的1％BSA中。丢弃来自井流体和冲洗板:用PBST 6次（300微升/孔），每次放置在摇床上10秒之前和在中间洗涤，一旦与卫生署2 O（300微升/孔），一个附加的6倍用PBST（300微升/孔），并两次用PBS（300微升/孔）。后细胞从板中除去，并且如果没有生物安全的担忧申请，程序可以被生物安全柜外进行。 </li><li>弃洗涤液。通过在纸巾轻敲板除去尽可能多的洗涤液成为可能。添加检测抗体（100μl/孔）。 </li><li>孵育板在120分钟或39°C。在该温育步骤中，准备按照制造商的说明书碱性磷酸酶溶液。使用前30分钟（即，除了检测抗体井后90分钟）稀释在PBST试剂。反转管轻轻混合试剂和在室温下孵育。 </li><li> 12后0分钟孵育，通过反演板块超额丢弃检测抗体。洗涤板6次用PBST（300微升/孔）。通过在纸巾轻敲板除去尽可能多的洗涤液成为可能。 </li><li>添加100μl/孔的碱性磷酸酶溶液（来自上述步骤3）。孵育板在室温45分钟。丢弃流体和用PBST洗涤各板6倍（300微升/孔）。 </li><li>制备下列制造商的说明（向量蓝AP底Ⅲ）底物溶液:稀释在100mM的Tris HCL pH值为8.2的缓冲试剂。吸管50微升/底物溶液很好。 </li><li>在室温下孵育直到蓝色开始发展（约30分钟）。丢弃液体和与卫生署2 O的丰富金额洗板除去底板以暴露膜，洗回水井，并允许板风干。 </li><li>阅读关于免疫斑点图像分析仪或ELISPOT读数器（表1），或手动板，使用立体显微镜。另外，保持板在黑暗中室温，直到读。由于反应基质的稳定性高，质量是保存了好几年。 注:细胞与抗原的浓度进行了优化，以避免井不可数点23,24,27,37。这是可能的不可数斑形成发生在不同的设置，或由于生物变异。如果是这样的话，细胞浓度应优化，以便获得一个可读斑点计数响应。 </li></ol> 7.板读取和数据分析<ol><li>执行分析具体根据蜂窝科技有限公司（CTL）的ELISPOT平板读取器引导件（表1）。后的斑点计数每个孔中得到的，计算重复间的平均值。每个抗原刺激的特异性免疫应答通过减去在从该抗原刺激孔的非刺激孔的斑点的平均数目进行计算。 </li></ol>

作者宣称，他们有没有竞争的财务权益。

IFN-γ的生产中的长期培养的ELISPOT分析一般是由于华健康在人类中，但这种反应在培养如何发展却知之甚少。是否华健康存在于循环和扩大在体外 ，或者如果来自分化的效应和TEM细胞进入医药文化期间华健康反应导致不得而知。然而，随着从人类样本的研究发现，从体外和长期培养的IFN-γ的ELISPOT分析CD4 + T细胞具有无关表位特异性，这意味着华健康响应不脱离效应细胞28发展。显然，响应的持续时间可以改变，但是如果病原体间隙来实现，最终都离体和TCM响应下降。此外，长期培养物的反应抗原引发后早期和长期测量（当效应器响应是低级）被示出为相关，这表明抗后早期和长循环华健康种群基因启动保持体内相关。在另一方面，在体外反应的程度似乎没有进行相关的记忆应答29的幅度。这些结果表明，长期培养的IFN-γELISPOT反应从体外扩增医药和效应响应的样品中的相关联的减弱，而不是效应细胞分化成华健康表型结果。 与牛，效应物和存储器的子集，以响应由长期的IFN-γELISPOT分析检测的相对贡献是未知的。最近的研究表明疫苗之间的相关性引起的长期培养的IFN-γ的ELISPOT反应和保护，防止随后的实验感染分枝牛 23-24。据报道，弱长期IFN-γ的ELISPOT反应接种与没有保护30的关联。无论生活和杀ED疫苗诱发类似的体外 IFN-γELISPOT反应，但接种卡介苗活强引起的长期培养的IFN-γ酶联免疫斑点比灭活疫苗制剂反应。有趣的是，活疫苗是保护而被杀的配方不能提供保护，防止挑战，有毒结核分枝杆菌31-32，33。 华健康反应的评估也是由PBMC整理这些细胞是可行的。华健康从PBMC直接富集，但是，需要昂贵的设备，训练有素的个人，是困难的，因为这些细胞不大量在血流中。 PBMC的长期培养提供中医富集在TEM和效应细胞，无需昂贵的设备;然而，因为这些T细胞群在体外扩展它们可以在体内记忆应答的代表以下。它可以访问的IFN-γ记忆应答（使用长期培养或TCM排序荷兰国际集团的策略）通过分析比ELISPOT其他如:细胞因子ELISA中34，细胞因子珠子阵列（CBA），细胞内染色（ICS），或细胞因子蛋白质阵列（CPA）。这些方法;然而，一般都是较不敏感的比ELISPOT分析35。 ELISPOT的一个优点是它的后不久其释放防止稀释在上清液和降解通过酶裂解或细胞因子的摄取通过其它细胞，以检测细胞因子的直接捕获能力。酶联免疫斑点试验检测单个细胞的细胞因子生产提供精确的结果，即使在低信噪比的情况下（ 即低特异性应答）35。另外，使用ICS，检测释放前的细胞因子，可能会导致细胞产生细胞因子的误识别（ 例如 ，由于之前或期间分泌过程翻译后调制）34。使用ICS测定以最小化细胞因子的分泌所用的转运抑制剂在抗原刺激（称为高尔基停止蛋白质）限制细胞刺激的持续时间，由于这些蛋白质最终影响细胞因子的产生器35的细胞毒性。 随着中说- CBA，CPA和ICS是用于同时测量多种细胞因子和/或用于确定细胞表面标记的表达有用的技术（ 即 ，使用ICS）。因此，这些方法可结合使用的IFN-γELISPOT测定36。虽然以前的牛结核病疫苗的有效性研究通过酶联免疫斑点法检测华健康响应，TCM反应通过其他技术检测可能会产生类似的结果。重要的是，酶联免疫斑点法是更便宜，更简单的CBA相比，CPA和ICS分析技术34来执行。 SFC的人工计数是替代自动计数，和ELISPOT板可以存储在RT长时间持续具有最小质量损失，之前或现货℃后ounting 37。 长期培养的IFN-γ的ELISPOT反应已应用于评估结核疫苗的反应时，以评估人的记忆应答，和牛14-16,31-32,33。中医响应也假定为在宿主发挥响应显著角色牛几个其它感染剂，如: 丝状支原体亚种丝状 42， 无形体 38和牛呼吸道合胞病毒39。潜在地，长期ELISPOT测定可以适于对其他动物物种，细胞因子和感染。这些更广泛的应用将是兽医免疫学应用特别有用由于目前有限的特定试剂的可用性。 中医的反应是防止感染的几个关键的，但感染/免疫接种后的早期测量它们（疾病结果的预测或疫苗的有效性）马y为繁琐。下离体和短抗原刺激条件的免疫应答的分析会更经常代表效应的反应，由于记忆和效应的响应的重叠，特别是在免疫学记忆形成的开始。在慢性疾病的情况下，其中的抗原负载是持续， 离体的反应将评估效应器细胞应答或记忆和效应细胞应答的组合。长期培养的IFN-γELISPOT测定，这里描述的，有可能使华健康响应而不是效应T细胞或组合的CD4 + T细胞应答的测定。总之，长期培养的IFN-γELISPOT试验提供了一个有价值的方法，用于估计的T细胞记忆应答，并已采用成功评估几个物种的存储器响应于各种感染剂12-16，20-25,29 ，31,33,34,38,39。

通过幼稚CD4 + T细胞的抗原识别后发育编程产生效应和记忆T细胞。幼稚的CD4 + T细胞分化成细胞因子产生细胞的分化，需要由先天免疫细胞，抗原呈递至T细胞，共刺激和产生偏振光的细胞因子产生的转录变化病原体识别。例如:抗原提呈细胞产生白细胞介素（IL）-12响应于细胞内的病原体，其中，连同抗原识别，通过信号转导和活化剂信令促进T细胞分化成T辅助细胞（Th1）细胞1,2-转录4（STAT4）和T-盒中的T细胞（T-bet的）表达，从而导致细胞活化，产生IL-2，克隆扩增和干扰素（IFN）-γ生产3,4-。如果感染被控制，高达95％的在膨胀阶段产生的T细胞被消除（ 即 ，收缩相）和记忆性T细胞依然存在，有时一生5。 Sallusto 等人 6，揭示了两个功能不同的记忆性T细胞的亚群在人类基于淋巴结归巢受体的表达。中央记忆T细胞（TCM）表达CC趋化因子受体（CCR）-7和CD45RO和主要位于次级淋巴器官的T细胞区域。中医具有有限的效应子功能，低激活阈值和保持高的IL-2产生和增殖能力。效应记忆T细胞（TEM）表达CD45RO，缺乏CCR7和显示受体归巢到外设或发炎的组织。效应细胞不表达任何CCR7或CD45RO但及时产生效应细胞因子，如IFN-γ，在抗原识别。 IFN-γ释放测定（IGRA）用于牛和人7结核病的诊断。 分枝杆菌是牛结核病（BTB）的主要代理，而人类CA结核病SES，主要是由结核杆菌引起的。用这些试验中，全血或外周血单核细胞（PBMC）的刺激是通过ELISA或通过检测细胞生产使用ELISPOT技术的IFN-γ测定分枝杆菌抗原为16至24小时，将上清液内IFN-γ的生产。作为短暂刺激期的结果（ 即 ，16至24小时），并快速的细胞因子生成， 离体测定法检测主要效应器和TEM响应。这已被证实通过细胞群中这些培养8-10流式细胞分析。 离体 IFN-γ的应答例行包括在结核病疫苗，包括那些用于评估由牛应答的免疫应答面板的评价。最有效的牛结核病疫苗诱导特异性IFN-γ的反应，而不是诱导IFN-γ应答所有的疫苗都protectiv即此外，IFN-γ通过接种疫苗引起，如感染前测定的水平，并不一定关联与保护。例如，不同的BCG菌株可以具有不同的能力，以诱导体外 IFN-γ应答，尽管类似的保护水平11。因此， 体外 IGRAs是结核病的诊断和疫苗的访问有价值的免疫原性;然而，它们作为疫苗功效的预测的使用是有限的。中医响应疫苗接种，在另一方面，可用于预测疫苗功效，如在非人灵长类12,13，结核小鼠14和疟疾在人类15,16证实猴免疫缺陷病毒（SIV）感染。 后导致病原体清除有效的免疫应答，TCM保持和用相同的试剂提供保护，以最终第二感染。一个值得注意的例外情况是M.肺结核 INFE人类ction其中接收治疗的抗分枝杆菌治疗的患者易受再感染17,18。此外，在慢性感染治免疫记忆的事件，其中所述抗原刺激仍然存在，都不能很好地理解19。在慢性感染，例如人类免疫缺陷病毒（HIV）和结核病，一个显著华健康响应与一个有利的结果（ 例如 ，等待时间以结核病和亚临床疾病与HIV）20,21相关联。几项研究与小鼠和人已经表明，长期培养的IFN-γ的ELISPOT分析测量华健康响应16,20-22。用该测定中，外周血是在降低抗原为10至14天的浓度，使效应器响应达到峰值，然后减弱培养;促进华健康分化和扩大文化中。 长期培养的IFN-γ的ELISPOT分析也已在兽医使用研究，但应答细胞的表型已经难以评估由于缺乏关键的试剂，尤其是抗体对CCR7的。有效的牛结核病疫苗[ 如 。使用M的单剂量牛卡介苗（BCG），BCG其次是病毒抗原向量85A亚单位疫苗或减毒牛分枝杆菌 ΔRD1]引起接种后的长期培养的IFN-γELISPOT反应与保护的相关（ 即更低的分枝杆菌负担和降低TB-相关的病理）对随后的挑战与毒力M.牛 23,24。此外，抗原特异性IFN-γ分泌细胞的长期PBMC培养物中的数字是在12个月高，但减少在新生儿小牛接种卡介苗后24个月内，以保护检测交分枝程度相关牛挑战25。在这种情况下，将培养的IFN-γELISPOT是一个预测疫苗的有效性，提供优先候选疫苗的成本高，药效试验一指N重要工具。此外，培养的ELISPOT技术可以通过改变抗原或抗体为多种目的在不同的研究领域适于各种宿主，病原体和细胞因子。

<table><tbody><tr><td>Sodium citrate (dihydrate)</td><td>Various</td><td></td><td></td></tr><tr><td>Citric acid (monohydrate)</td><td>Various</td><td></td><td></td></tr><tr><td>Dextrose</td><td>Various</td><td></td><td></td></tr><tr><td>ELISPOT PVDF plate</td><td>Various</td><td></td><td></td></tr><tr><td>Vectastain ABC &amp;ndash; AP KIT Standard</td><td>Vector Laboratories</td><td>AK-5000</td><td></td></tr><tr><td>Vector Blue Alkaline Phosphatase Substrate Kit</td><td>Vector Laboratories</td><td>SK-5300</td><td></td></tr><tr><td>Ag85A</td><td>Lionex</td><td>LRP-0004.3</td><td>23, 24, 25, 37</td></tr><tr><td>TB 10.4</td><td>Lionex</td><td>LRP-0061.6</td><td>23, 24, 25, 37</td></tr><tr><td>PPDb</td><td>Prionics AG</td><td>7600055</td><td></td></tr><tr><td>rESAT-6:CFP10</td><td>Kind gift Dr. Minion, Iastate</td><td></td><td>23, 24, 25, 27,&amp;nbsp; 37</td></tr><tr><td>Mouse anti-bovine IFN-&amp;gamma; Clone CC302</td><td>Serotec</td><td>MCA1783B</td><td>23, 24, 25, 27,&amp;nbsp; 37</td></tr><tr><td>Mouse anti-bovine IFN-&amp;gamma; Biotinylated Clone CC330</td><td>Serotec</td><td>MCA2112</td><td>23, 24, 25, 27,&amp;nbsp; 37</td></tr><tr><td>Mouse anti-bovine CD4 Clone CC8</td><td>Serotec</td><td>MCA1653GA</td><td></td></tr><tr><td>Mouse anti-bovine CD45RO Clone IL116A</td><td>Serotec</td><td>MCA2434GA</td><td></td></tr><tr><td>Rat anti-human CCR7 Clone 3D12</td><td>Abcam</td><td>ab95665</td><td></td></tr><tr><td>Mouse anti-bovine IFN-&amp;gamma;-Pe Clone CC302</td><td>Serotec</td><td>MCA1783PE</td><td></td></tr><tr><td>Goat anti- mouse IgG2a- Alexa Fluor 350</td><td>Invitrogen</td><td>A-21130</td><td></td></tr><tr><td>Goat anti-mouse IgG3 APC-CY7</td><td>SouthernBiotechnology</td><td>1080-193</td><td></td></tr><tr><td>Goat anti-rat IgG-APC</td><td>Invitrogen</td><td>A10540</td><td></td></tr><tr><td>BD Cytofix/Cytoperm&amp;trade;</td><td>BD Biosciences</td><td>554714</td><td></td></tr><tr><td>BrefeldinA</td><td>Sigma-Aldrich</td><td>B7651</td><td></td></tr><tr><td>Pokeweed Mitogen</td><td>Sigma-Aldrich</td><td>L8777</td><td></td></tr><tr><td>Recombinat human Interleukin 2</td><td>Sigma-Aldrich</td><td>I7908</td><td></td></tr></tbody></table>

application of long-term cultured interferon-&#947; enzyme-linked immunospot assay for assessing effector and memory t cell responses in cattle

通过幼稚细胞的以下抗原识别发育编程产生效应和记忆T细胞。如果感染被控制到95％的过程中的膨胀阶段中产生的T细胞被消除（ 即 ，收缩相位）和记忆T细胞保持，有时一生。在人类中，两个功能不同的记忆性T细胞的亚群基于淋巴结归巢受体的表达已被描述。中央记忆T细胞表达CC趋化因子受体7和CD45RO和主要位于次级淋巴器官的T细胞区域。效应记忆T细胞表达CD45RO，缺乏与淋巴细胞归巢相关的外设或发炎组织CCR7和显示受体。效应T细胞不表达任一CCR7或CD45RO但是当与抗原产生效应细胞因子，如干扰素γ相遇。干扰素γ释放分析被用于牛和人类结核的诊断和检测主要效应和效应记忆T细胞应答。由CD4 + T细胞接种中央记忆T细胞应答，在另一方面，可用于预测疫苗功效，其表现与猴免疫缺陷病毒感染的非人类灵长类动物，肺结核在小鼠和疟疾在人类。与小鼠和人类以及对牛未公布的数据的一些研究，已经表明，干扰素γELISPOT分析测量中央记忆T细胞应答。用该测定中，外周血单核细胞是在降低抗原浓度为10〜14天（长期培养物），从而允许执行器响应达到峰值，然后减弱培养;促进中央记忆T细胞分化和扩大文化中。

大约一个月后气溶胶感染分枝牛枝原体 （10 4菌落形成单位），PBMC中从感染的（N = 8）和对照组动物（每组8）分别在抗原和IL-2的13天的存在下进行培养。华健康反应发展感染后，用IFN-γ的ELISPOT测定法测定。代表TCM（在APC细胞的存在或不存在）和离体 IFN-γ从感染的动物（三只动物）和非感染的动物（一只动物）示于图1中的ELISPOT反应。成功的长期IFN-γ的ELISPOT测定的结果的细胞中产生IFN-γ（斑点形成细胞，SFC）下刺激条件和几乎没有来自未感染的动物的响应的和非刺激的条件下进行。此外，强烈的T细胞反应应该发生在响应PWM。以M.具体回应CFP10抗原刺激: 牛被PPD-B或雷沙特-6进行评估。 如图1，强大的中医体外反应PPD-B和雷沙特-6:CFP10从三个检测M.牛感染的动物。最少没有传统中医和体外的反应是从控制动物检测。此外，被要求由受感染的动物最佳华健康响应的APC的存在下，这表现在大大减少的响应由在不存在自体的APC的培养长期细胞。通过从分枝的PBMC的IFN-γ反应杆菌感染和未感染的动物都在图2中呈现。SFC的来自每个动物的数目计算为SFC的平均数目两份样品中响应于PPD-B减到单独的培养基的各自的响应。响应被估计为10 6个细胞。反应差异（P &lt;0.05）根据感染情况，有无装甲运兵车和培养持续时间（ 即 ，长期培养与体外培养）的。 <table border="0" cellpaddin克="0"CELLSPACING ="0"&gt; <colgroup><col /><col /></colgroup><tbody><tr><td colspan="2">采集板的图像</td></tr><tr><td> 1。 </td><td>打开机</td></tr><tr><td> 2。 </td><td>打开"免疫捕获"6.3版软件。 </td></tr><tr><td> 3。 </td><td>第1步:选择板型。 </td></tr><tr><td> 4。 </td><td>第2步:负载板。 </td></tr><tr><td> 5。 </td><td>第3步:选择扫描选项。 </td></tr><tr><td> 6。 </td><td>第4步:开始扫描。 </td></tr><tr><td> 7。 </td><td>获得板全貌图像。 </td></tr><tr><td> 8。 </td><td>排出板。 </td></tr><tr><td> 9。 </td><td>退出"免疫捕捉"软件。 </td></tr><tr><td colspan="2">计数斑点形成单位</td></tr><tr><td> 1。 </td><td>打开"免疫点捕捉"5.0版软件。 </td></tr><tr><td> 2。 </td><td>选择objec<html> T类:正常。 </td></tr><tr><td> 3。 </td><td>选择计数模块:智能计数。 </td></tr><tr><td> 4。 </td><td>第1步:载重板。 </td></tr><tr><td> 5。 </td><td>第2步:定义计数参数:现货识别测试精度上井的不同点。 </td></tr><tr><td> 6。 </td><td>开始自动计数。 </td></tr><tr><td colspan="2">质量控制</td></tr><tr><td> 1。 </td><td>打开"免疫斑点捕捉"5.0版软件。 </td></tr><tr><td> 2。 </td><td>选择计数模块:质量控制。 </td></tr><tr><td> 3。 </td><td>第1步:载重板。 </td></tr><tr><td> 4。 </td><td>第2步:单独分析高亮井。 </td></tr><tr><td> 5。 </td><td>完成质量控制。 </td></tr></tbody></table> 表1 - AutoImmun Diagnostika ELISPOT读取器图像采集和细胞计数的过程。 ove_content"FO:保持together.within页="总是"&gt; <img alt="图1" src="/files/ftp_upload/52833/52833fig1.jpg" /> 从长期培养的IFN-γELISPOT测定孔的图1的图像。培养的IFN-γ的ELISPOT测定法approximatelyone一个月毒性分枝挑战之后执行牛 （三只动物）。非感染的动物作为对照（一个动物）通过刺激的PBMC用重组Ag85A双（1微克/毫升），TB10.4（1微克/毫升）的鸡尾酒生成长期细胞系，雷沙特-6: CFP10（1微克/毫升）和PPD-B（5微克/毫升）13天，然后转移到ELISPOT板中的自体的APC的存在下或不存在。短期细胞组成的分离外周血单个核细胞在第13天，直接铺在ELISPOT板。长期和短期的细胞用PPD-B（10微克/毫升），雷沙特-6:CFP10的（1微克/毫升），单独培养基或美洲商陆有丝分裂原（5＆＃956;微克/毫升）处理24小时。 <img alt="图2" src="/files/ftp_upload/52833/52833fig2.jpg" /> 从长期培养的IFN-γ的ELISPOT反应至M.图2代表性的结果牛分枝杆菌纯化的蛋白衍生物（PPD-B）培养的IFN-γELISPOT测定法用有毒力的分枝挑战后approximatelyone月进行牛枝原体 （8只动物）。非感染的动物作为对照（通过刺激的PBMC用重组Ag85A双的鸡尾酒（1微克/毫升），TB10.4（1微克/毫升），分别生成8个动物的长期细胞雷沙特-6:CFP10（1微克/毫升）和PPD-B（5微克/毫升）13天，然后转移到ELISPOT板中的自体的APC的存在下或不存在。短期细胞包括外周血单个核细胞的分离第13天，并直接镀到ELISPOT板。长期与笑室温长期刺激细胞使用PPD-B（10微克/毫升）或单独的培养基24小时。从每只动物（SFC / 10 6个细胞）特异性应答表示为响应于PPD-B（平均一式两份样品的）减去响应于单独的培养基。

Watch this Scientific Journal Video about Application of Long-term cultured Interferon-ÃŽÂ³ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle at JoVE.com

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

长期培养的干扰素γ酶联免疫斑点法被用作中央记忆应答的量度并且与保护性抗分枝杆菌疫苗的反应。用该测定中，外周血单核细胞刺激与分枝杆菌抗原和IL-2 14天，使分化和扩展中央记忆T细胞。

长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

application-long-term-cultured-interferon-enzyme-linked-immunospot

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JoVE Journal

Immunology and Infection

12.1K 次观看.  United States Department of Agriculture. 长期培养的干扰素γ酶联免疫斑点法被用作中央记忆应答的量度并且与保护性抗分枝杆菌疫苗的反应。用该测定中，外周血单核细胞刺激与分枝杆菌抗原和IL-2 14天，使分化和扩展中央记忆T细胞。 

视频: Application of Long-term cultured Interferon-γ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.

Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

酶联免疫斑点法（ELISPOT）：TH - 1细胞对微生物抗原的免疫应答的定量

Adaptive immunity is an important component to clearance of intracellular pathogens.  The ability to detect and quantify these responses in humans is an ...

科研

免疫与感染

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

使用γ-干扰素酶酶联免疫检测的特点人类乳头状瘤病毒的新型T-细胞表位

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

荧光目标阵列的T细胞反应评价中的应用<em&gt;在体内</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens. &#160;

Novel generations of functional assays such as gamma interferon (IFN-&#947;) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

的IFN-γ酶联免疫斑点检测，以评估水痘 - 带状疱疹病毒特异性细胞免疫继脐血移植的发展

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

的分离，分化和人类的定量分泌抗体的B细胞从血液:酶联免疫斑点作为体液免疫的功能读出

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

优化干扰素伽隆弹性体检测在几内亚猪模型接种疫苗后 t 细胞反应

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity.
Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes.
The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-&#947; and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-&#947; secretion assessed using ELISA showed a significantly higher titer (**p &#60; 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.

The methodology describes the generation of bovine monocyte-derived dendritic cells (MoDCs) and their application for the in vitro evaluation of antigenic candidates during the development of potential veterinary vaccines in cattle.

使用牛单核细胞来源的树突状细胞测定疫苗免疫原性

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...

ELISPOT Assay: Detection of IFN-&#947; Secreting Splenocytes

Source: Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201
ELISPOT is a standardized, reproducible assay used to detect cellular immune responses. The assay utilizes an enzyme-linked immunosorbent assay (ELISA)- based method to detect single-cell immune responses which can be visualized by spots, hence the name ELISPOT. ELISPOT was first described in 1983, by Czerkinsky, as a method of enumerating the number of B cell hybridomas producing antigen-specific immunoglobulins (1). The same group further developed the assay to measure the frequency of cytokine producing T lymphocytes. Now ELISPOT has become a gold standard for measuring antigen-specific T cell immunity in clinical trials and vaccine candidates. For example, after vaccination or during an infection, plasma cells and memory B cells secrete antibodies that provide protection. Typically, these B cell responses are assessed by measuring serum titers of antigen-specific antibodies. However, this type of analysis, typically measured by ELISA, may not include memory B cells, which can be present even in the absence of detectable serum antibody levels. Furthermore, it has been well-established that circulating memory B cells are important for the rapid and protective antibody response observed following pathogen re-exposure, thus it is critical to be able to detect these cells. Therefore, to clearly assess antigen-specific memory B-cell responses, both ELISA and ELISPOT should be used (2).
ELISPOT assay uses a plate containing membrane-lined wells that are coated with antibodies in order to capture secreted proteins of interest. Then, the plate is loaded with cells and stimuli to induce protein production. The secreted proteins are captured by the antibodies coated on the surface. After appropriate incubation time, cells are removed and the secreted molecule is detected by using a biotinylated antibody that is specific for a different epitope, as compared to the capture antibody. Next, streptavidin peroxidase is added, followed by the addition of a substrate that allows the detection of the spots (Figure 1). The strength of this assay is that it allows one to quantitate the number of cells producing the protein of interest. Importantly, one can assess if there are changes in the total number of cells producing a specific protein or if individual cells within a population are producing more protein. Moreover, it can provide information regarding kinetics and can be used to assess overall immune activation (mitogen stimulation) relative to antigen-specific responses (antigen simulation). The ELISPOT assay will permit the detection one activated cell amongst 300,000 cells following mitogenic or antigen-specific activation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10497/10497fig1.jpg" /> 
Figure 1: ELISPOT protocol overview.
The major advantages of this assay are its- a. Simplicity- the protocol is relatively simple and straightforward. It does not require technical expertise, b. Sensitivity- it permits the detection of immune cells at the single cell level and requires very few cells compared to other methods such as flow cytometry, c. Functionality- it provides quantitative data regarding immune function.
This lab exercise demonstrates the ELISPOT protocol for detection of IFN-&#947; secreting-splenocytes, but as mentioned above this assay can also be used to assess antibody secretion by B cells (3).

ELISPOT Assay: Detection of IFN-&amp;#947; Secreting Splenocytes

ELISPOT 分析:检测 IFN-- 分泌性细胞

Source: Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum ...

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Assay for the Identification of Interferon-Gamma Secretion in Murine Lymphoid Organs

Source: Mazet, J. M., et al. <a href="https://app.jove.com/v/59819">Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection.</a> J. Vis. Exp. (2019).
This video demonstrates an imaging method to visualize in situ interferon-gamma (IFN-&#947;) secretion in the mouse spleen. Following bacterial infection and the induction of IFN-&#947; accumulation inside T cells and natural killer cells, the spleen is harvested from the mouse. Upon obtaining tissue sections, immunostaining is performed to visualize IFN-&#947; inside the cells.

小鼠淋巴器官中干扰素-γ 分泌鉴定的测定

Source: Mazet, J. M., et al. Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection. J. Vis. Exp. ...

长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

摘要

探索更多视频

此视频中的章节

酶联免疫斑点法（ELISPOT）：TH - 1细胞对微生物抗原的免疫应答的定量

使用γ-干扰素酶酶联免疫检测的特点人类乳头状瘤病毒的新型T-细胞表位

在体外试验，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

荧光目标阵列的T细胞反应评价中的应用

的IFN-γ酶联免疫斑点检测，以评估水痘 - 带状疱疹病毒特异性细胞免疫继脐血移植的发展

的分离，分化和人类的定量分泌抗体的B细胞从血液:酶联免疫斑点作为体液免疫的功能读出

优化干扰素伽隆弹性体检测在几内亚猪模型接种疫苗后 t 细胞反应

使用牛单核细胞来源的树突状细胞测定疫苗免疫原性

ELISPOT 分析:检测 IFN-- 分泌性细胞

小鼠淋巴器官中干扰素-γ 分泌鉴定的测定

长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

摘要

探索更多视频

此视频中的章节

酶联免疫斑点法（ELISPOT）：TH - 1细胞对微生物抗原的免疫应答的定量

使用γ-干扰素酶酶联免疫检测的特点人类乳头状瘤病毒的新型T-细胞表位

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

荧光目标阵列的T细胞反应评价中的应用

的IFN-γ酶联免疫斑点检测，以评估水痘 - 带状疱疹病毒特异性细胞免疫继脐血移植的发展

的分离，分化和人类的定量分泌抗体的B细胞从血液:酶联免疫斑点作为体液免疫的功能读出

优化干扰素伽隆弹性体检测在几内亚猪模型接种疫苗后 t 细胞反应

使用牛单核细胞来源的树突状细胞测定疫苗免疫原性

ELISPOT 分析:检测 IFN-- 分泌性细胞

小鼠淋巴器官中干扰素-γ 分泌鉴定的测定

在体外试验，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响